Modified ribonucleosides abound in RNA, yet their biological and structural significance are known in only a few cases. The most abundant such nucleoside is pseudouridine in which the uracil ring is post- transcriptionally rotated 2 positios to create a new linkage with the ribose. Despite being present in all studied organisms, the function of pseudouridine is known in only a few instances related to the structure of tRNA and rRNA. Recent results suggest that pseudouridylation may also play an important regulatory function but the mechanisms by which it mediates such effects are not known. In this application, work is described that will elucidate, for the first time, the role of pseudouridylatin in developmental biology using the important human parasite, Toxoplasma gondii, as the experimental system. Upon infection of a new host, Toxoplasma gondii initially expands as a systemic, fast-growing tachyzoite, after which it differentiates into a slow-growing, encysted bradyzoite to produce a chronic infection. The work proposed here exploits our surprising discovery that a pseudouridine synthase (TgPUS1) is crucial for the asexual differentiation of Toxoplasma, thereby affording the opportunity to determine the role of pseudouridylation in a complex biological process. The first aim proposed will use a novel method to identify essentially all pseudouridylation events in the two asexual forms of wild type Toxoplasma. Preliminary results show the power of this method with the successful identification of many expected pseudouridines (e.g., in conserved positions in rRNA), as well as several such modifications that are developmentally regulated. In aim 2, TgPUS1 mutants will be used to determine which pseudouridylation events are dependent on this enzyme for their creation. Again, preliminary results show the surprising and exciting discovery of TgPUS1-dependent pseudouridines in several mRNAs establishing the feasibility of the approach and confirming the biochemical activity of the TgPUS1. Aim 3 will develop a method for manipulating specific pseudouridylation sites on individual mRNA species in Toxoplasma tachyzoites as a prelude to eventual testing for the mechanism by which such TgPUS1-dependent pseudouridines play a key role in tachyzoite-to-bradyzoite differentiation. Successful execution of the work proposed here will provide crucial information on the asexual development of an important human parasite as well as giving the first insight into how the most commonly modified ribonucleoside can play a key regulatory role in development.